CN101301383B

CN101301383B - Quality control method of formulation for clearing wind-heat of children

Info

Publication number: CN101301383B
Application number: CN2008101163104A
Authority: CN
Inventors: 陈致慜; 李春雷; 霍志金; 李红梅; 刘宇
Original assignee: HANDAN MOLUODAN PHARMACEUTICAL CO Ltd
Current assignee: HANDAN PHARMACEUTICAL CO., LTD.
Priority date: 2008-07-08
Filing date: 2008-07-08
Publication date: 2011-12-28
Anticipated expiration: 2028-07-08
Also published as: CN101301383A

Abstract

The invention discloses a method for controlling the quality of children Fengreqing mixture. The method adopts the high performance liquid chromatography to measure the content of geniposide contained in the children Fengreqing mixture and identify a plurality of effective components; the method achieves higher standards according to national standards of the National Institute For the Control of Pharmaceutical and Biological Products, so that the method has the advantages of being easily operated, saving measuring costs and lowering the toxicity of the reagent.

Description

The detection method of formulation for clearing wind-heat of children

Technical field

The present invention relates to a kind of detection method of pharmaceutical composition, particularly the detection method of formulation for clearing wind-heat of children.

Background technology

It is paediatrics common disease, frequently-occurring disease clinically that cat fever, acute simple tonsillitis or suppurative tonsillitis merge fever (diseases caused by external factors, newborn pretty young woman are suppurated to hold concurrently and had a fever).The medicine of generally acknowledging both at home and abroad is a microbiotic at present, and is domestic many with the penicillin intramuscular injection, and penicillin resistance is growing, and the part infant can not be accepted the treatment of this medicine again because of allergy, and it is also very painful that a lot of infants are accepted this medicine.Formulation for clearing wind-heat of children is by the oral liquid of flavour of a drug through being processed into such as honeysuckle, the capsule of weeping forsythia, Radix Isatidis, peppermint, radix bupleuri, great burdock achene, ching-chieh, gypsum, the root of large-flowered skullcap, cape jasmine, balloonflower roots, has relieving the exterior syndrome with drugs pungent in flavor and cool in property, clearing heat and detoxicating, the effect of cough-relieving relieve sore throat is used for children's wind-heat cold, heating, cough, expectoration, nasal obstruction runny nose, red swelling and pain of throat; For the quality of strictness control formulation for clearing wind-heat of children, be necessary to study the detection method of a cover formulation for clearing wind-heat of children.

Summary of the invention

One object of the present invention is the detection method of open formulation for clearing wind-heat of children.

The present invention seeks to be achieved through the following technical solutions:

The detection method of formulation for clearing wind-heat of children comprises one or more in following discriminating and/or the content assaying method:

Differentiate:

(1) gets formulation for clearing wind-heat of children 10-50ml, add watery hydrochloric acid adjust pH to 1～2, put in the separating funnel, extract 1-3 time with solvent ether, 60～90 ℃ of sherwood oils or methenyl choloride, each 10-30ml, discard solvent ether, 60～90 ℃ of sherwood oils or methenyl choloride liquid, use ethyl acetate extraction 2-4 time again, each 10-30ml, combined ethyl acetate liquid, water bath method, residue add methyl alcohol 1-5ml dissolving, as need testing solution; Other gets burdock glycosides reference substance, adds methyl alcohol and makes the solvent that every 1ml contains 1-5mg, in contrast product solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw reference substance solution 5 μ l, need testing solution 10 μ l, put respectively on same silica gel g thin-layer plate, with methenyl choloride-methanol-water is that 10-50: 1-10: 1-5 is a developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing at 105 ℃; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color;

(2) get formulation for clearing wind-heat of children 10-50ml, add watery hydrochloric acid adjust pH to 1～2, put in the separating funnel, extract 1-3 time with solvent ether, 60～90 ℃ of sherwood oils or methenyl choloride, each 10-30ml, discard solvent ether, 60～90 ℃ of sherwood oils or methenyl choloride liquid, use ethyl acetate extraction 2-4 time again, each 10-30ml, combined ethyl acetate liquid, water bath method, residue add methyl alcohol 1-5ml dissolving, as need testing solution; Other gets the scutelloside reference substance, adds methyl alcohol and makes solution that every ml contains 0.5-5mg product solution in contrast; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw reference substance solution 5 μ l, need testing solution 2 μ l, put respectively on same silica gel g thin-layer plate, with ethyl acetate-butanone-formic acid-water is that 3-10: 1-5: 0.1-5: 0.1-5 is a developping agent, launch, take out, dry, spray is with 1% ferric trichloride ethanolic solution; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color;

(3) get formulation for clearing wind-heat of children 10-50ml, inject in the D101 type macroporous adsorptive resins of the internal diameter 1.5cm, the high 25cm of post that have handled well, use the speed wash-out of the distilled water of 100-500ml earlier, discard eluent with 1-5ml/min, use the ethanol elution of 50-95% again, the collection eluent, shakes up to scale in the 100ml measuring bottle, filters, get subsequent filtrate 20-80ml and put evaporating dish, evaporate to dryness, residue add methyl alcohol 1-5ml dissolving, as need testing solution; Other gets the Gardenoside reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5-5mg, in contrast product solution; Test according to an appendix VIB of Chinese Pharmacopoeia version in 2005 thin-layered chromatography, draw need testing solution 10 μ l, reference substance solution 5 μ l, put respectively on same silica gel g thin-layer plate, with methenyl choloride-methyl alcohol-acetonitrile-ammoniacal liquor is that 1-10: 1-5: 0.1-1: 0.1-1 is a developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and 105 ℃ to be heated to the spot colour developing clear; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;

(4) get formulation for clearing wind-heat of children 10-50ml, with ammonia solution adjust pH to 10, with solvent ether, 60～90 ℃ of sherwood oils or chloroform extraction 1-3 time, each 10-20ml, divide and to get solvent ether, 60～90 ℃ of sherwood oils or methenyl choloride liquid, evaporate to dryness, residue add methyl alcohol or ethanol 1-5ml makes dissolving, as need testing solution; Other gets capsule of weeping forsythia control medicinal material 1-5g and adds water 50-100ml, decocts 10-60 minute, filters, and filtrate is concentrated into 10-50ml, prepares medicinal material solution in contrast with method; Test according to an appendix VIB of Chinese Pharmacopoeia version in 2005 thin-layered chromatography; Drawing each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, is that 2-10: 2-10: 1-5 is a developping agent with methenyl choloride-ethyl acetate-methyl alcohol, launch, take out, dry, spray is with 5% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing at 105 ℃; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of identical color;

(5) get formulation for clearing wind-heat of children 10-50ml, add water 5-20ml, shake up, add watery hydrochloric acid and regulate pH value to 1～2, centrifugal 10 minutes, divide get supernatant solubilizer ether, 60～90 ℃ of sherwood oils or methenyl choloride, evaporate to dryness is extracted in ethyl acetate 10-30ml jolting 2-3 time, residue solubilizer ether, 60～90 ℃ of sherwood oils or methenyl choloride, ethyl acetate 1-5ml make dissolving, as need testing solution; Other gets the chlorogenic acid reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.1-1mg, in contrast product solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw each 2 μ l of above-mentioned two kinds of solution, put respectively on same polyamide film, with ethyl acetate-methyl alcohol-formic acid is that 5-10: 0.1-5: 0.1-5 is a developping agent, launch, take out, dry, put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color;

(6) get formulation for clearing wind-heat of children 10-50ml, use solvent ether, the 365nm sherwood oil, methenyl choloride, ethyl acetate or water-saturated n-butanol jolting are extracted 1-3 time, each 10-50ml, merge solvent ether, 60～90 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol liquid, wash with 2% sodium hydroxide solution 10-30ml, use solvent ether again, 60～90 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol washing 2 times, each 30ml, discard washing lotion, solvent ether, 60～90 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol solution evaporate to dryness, residue adds methyl alcohol 1-5ml makes dissolving, as need testing solution; Other gets radix bupleuri control medicinal material 1-5g, adds water 50-100ml, heats little boiling 1-5 hour, and filtrate is shone medicinal material solution in pairs with legal system; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw above-mentioned need testing solution 10 μ l, control medicinal material solution 5 μ l, put respectively on same silica gel g thin-layer plate, with methenyl choloride-methanol-water is that 10-50: 5-30: 0.5-10 is a developping agent, launch, taking-up is dried, and spray is with 40% sulfuric acid solution of 2% paradime thylaminobenzaldehyde, and 60-100 ℃ to be heated to the spot colour developing clear; Put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence principal spot of same color;

(7) get formulation for clearing wind-heat of children 10-50ml, put in the separating funnel, solubilizer ether, 60～90 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol 10-30ml joltings are extracted 1-3 time, divide and get solvent ether, 60～90 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol liquid, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the aurantiin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.1-1mg, in contrast product solution; Test according to an appendix VIB of Chinese Pharmacopoeia version in 2005 thin-layered chromatography, draw above-mentioned need testing solution 10 μ l, reference substance solution 5 μ l, put respectively on same silica gel g thin-layer plate, with ethyl acetate-butanone-formic acid-water is that 2-10: 1-5: 0.1-3: 0.1-5 is a developping agent, launch, taking-up is dried, and spray is with 2% aluminium choride ethanolic solution, 105 ℃ of heating several minutes, put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color.

Assay:

Precision is measured formulation for clearing wind-heat of children 10-50ml, inject in the D101 type macroporous adsorptive resins of the internal diameter 1.5cm, the high 25cm of post that have handled well, use the speed wash-out of the distilled water of 100-500ml earlier with 1-5ml/min, discard eluent, use the ethanol elution of 50-95% again, collect eluent in the 100ml measuring bottle to scale, shake up, filter, get subsequent filtrate, promptly get need testing solution; Precision takes by weighing through the Gardenoside reference substance 10mg of phosphorus pentoxide drying under reduced pressure more than 12 hours, puts in the 25ml measuring bottle, adds dissolve with methanol to scale, shakes up, precision is measured 1ml, puts in the 10ml measuring bottle, adds methyl alcohol to scale, shake up, contain Gardenoside 40 μ g among every 1ml, promptly get reference substance solution; With octadecylsilane chemically bonded silica is filling agent, and acetonitrile-water is that 5-30: 95-70 is a moving phase, detects wavelength 238nm, and number of theoretical plate calculates by the Gardenoside peak should be not less than 2000; According to high effective liquid chromatography for measuring, accurate respectively reference substance solution and each 10 μ l injection liquid chromatograph of need testing solution drawn measured, that is, every 1ml formulation for clearing wind-heat of children contains cape jasmine with Gardenoside C ₁₇H ₂₄O ₁₀Meter must not be less than 0.16mg.

The detection method of formulation for clearing wind-heat of children is preferably as follows one or more in discriminating and/or the content assaying method:

Differentiate:

(1) gets formulation for clearing wind-heat of children 10ml, add watery hydrochloric acid adjust pH to 2, put in the separating funnel, extract 2 times with solvent ether, 60 ℃ of sherwood oils or methenyl choloride, each 10ml, discard solvent ether, 60 ℃ of sherwood oils or methenyl choloride liquid, use ethyl acetate extraction again three times, each 20ml, combined ethyl acetate liquid, water bath method, residue add methyl alcohol 1ml dissolving, as need testing solution; Other gets burdock glycosides reference substance, adds methyl alcohol and makes the solvent that every 1ml contains 1mg, in contrast product solution; Test according to an appendix VIB of Chinese Pharmacopoeia version in 2005 thin-layered chromatography, draw reference substance solution 5 μ l, need testing solution 10 μ l, put respectively on same silica gel g thin-layer plate, with methenyl choloride-methanol-water be 10: 10: 1 be developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing at 105 ℃; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color;

(2) get formulation for clearing wind-heat of children 50ml, add watery hydrochloric acid adjust pH to 1, put in the separating funnel, extract 2 times with solvent ether, 90 ℃ of sherwood oils or methenyl choloride, each 30ml, discard solvent ether, 90 ℃ of sherwood oils or methenyl choloride liquid, use ethyl acetate extraction again three times, each 20ml, combined ethyl acetate liquid, water bath method, residue add methyl alcohol 5ml dissolving, as need testing solution; Other gets the scutelloside reference substance, adds methyl alcohol and makes solution that every ml contains 5mg product solution in contrast; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw reference substance solution 5 μ l, need testing solution 2 μ l, put respectively on same silica gel g thin-layer plate, with ethyl acetate-butanone-formic acid-water is 10: 1: 5: 0.1 is developping agent, launch, take out, dry, spray is with 1% ferric trichloride ethanolic solution; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color;

(3) get formulation for clearing wind-heat of children 10ml, inject in the D101 type macroporous adsorptive resins of the internal diameter 1.5cm, the high 25cm of post that have handled well, use the speed wash-out of the distilled water of 100ml earlier, discard eluent with 1ml/min, use 50% ethanol elution again, the collection eluent, shakes up to scale in the 100ml measuring bottle, filters, get subsequent filtrate 40ml and put evaporating dish, evaporate to dryness, residue add methyl alcohol 3ml dissolving, as need testing solution; Other gets the Gardenoside reference substance, adds methyl alcohol and makes the solution that every 1ml contains 2.5mg, in contrast product solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw need testing solution 10 μ l, reference substance solution 5 μ l, put respectively on same silica gel g thin-layer plate, with methenyl choloride-methyl alcohol-acetonitrile-ammoniacal liquor is 5: 2: 0.5: 0.6 is developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and 105 ℃ to be heated to the spot colour developing clear; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;

(4) get formulation for clearing wind-heat of children 20ml, with ammonia solution adjust pH to 10, with solvent ether, 70 ℃ of sherwood oils or chloroform extraction 2 times, each 20ml divides and gets solvent ether, 70 ℃ of sherwood oils or methenyl choloride liquid, evaporate to dryness, residue adds methyl alcohol or ethanol 2ml makes dissolving, as need testing solution; Other gets capsule of weeping forsythia control medicinal material 2g and adds water 90ml, decocts 20 minutes, filters, and filtrate is concentrated into 20ml, prepares medicinal material solution in contrast with method; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography; Draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with methenyl choloride-ethyl acetate-methyl alcohol be 3: 9: 3 be developping agent, launch, take out, dry, spray is with 5% ethanol solution of sulfuric acid, 105 ℃ be heated to spot develop the color clear; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of identical color;

(5) get formulation for clearing wind-heat of children 40ml, add water 20ml, shake up, add watery hydrochloric acid and regulate pH value to 1, centrifugal 10 minutes, divide and get supernatant solubilizer ether, 80 ℃ of sherwood oils or methenyl choloride, ethyl acetate 30ml jolting extraction 3 times, divide and to get solvent ether, 80 ℃ of sherwood oils or methenyl choloride, acetic acid ethyl fluid, evaporate to dryness, residue solubilizer ether, 80 ℃ of sherwood oils or methenyl choloride, ethyl acetate 4ml make dissolving, as need testing solution; Other gets the chlorogenic acid reference substance, adds methyl alcohol and makes the solution that every 1ml contains 1mg, in contrast product solution; According to the test of an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw each 2 μ l of above-mentioned two kinds of solution, put respectively on same polyamide film, with ethyl acetate-methyl alcohol-formic acid be 10: 0.5: 4 be developping agent, launch, take out, dry, put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color;

(6) get formulation for clearing wind-heat of children 25ml, use solvent ether, 65 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol jolting are extracted 2 times, each 25ml, merge solvent ether, 65 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol liquid, wash with 2% sodium hydroxide solution 20ml, use solvent ether again, 65 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol washing 2 times, each 30ml, discard washing lotion, solvent ether, 65 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol solution evaporate to dryness, residue adds methyl alcohol 5ml makes dissolving, as need testing solution; Other gets radix bupleuri control medicinal material 5g, adds water 70ml, heats little boiling 2 hours, and filtrate is shone medicinal material solution in pairs with legal system; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw above-mentioned need testing solution 10 μ l, control medicinal material solution 5 μ l, put respectively on same silica gel g thin-layer plate, with methenyl choloride-methanol-water be 50: 15: 5 be developping agent, launch, taking-up is dried, and spray is with 40% sulfuric acid solution of 2% paradime thylaminobenzaldehyde, and 60 ℃ to be heated to the spot colour developing clear; Put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence principal spot of same color;

(7) get formulation for clearing wind-heat of children 50ml, put in the separating funnel, solubilizer ether, 90 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol 30ml joltings are extracted 3 times, divide and get solvent ether, 90 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol liquid, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the aurantiin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 1mg, in contrast product solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw above-mentioned need testing solution 10 μ l, reference substance solution 5 μ l, put respectively on same silica gel g thin-layer plate, with ethyl acetate-butanone-formic acid-water is 10: 1: 3: 0.1 is developping agent, launch, taking-up is dried, and spray is with 2% aluminium choride ethanolic solution, 105 ℃ of heating several minutes, put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color.

Assay:

Precision is measured formulation for clearing wind-heat of children 10ml, inject in the D101 type macroporous adsorptive resins of the internal diameter 1.5cm, the high 25cm of post that have handled well, use the speed wash-out of the distilled water of 100ml earlier with 1ml/min, discard eluent, use 50% ethanol elution again, collect eluent in the 100ml measuring bottle to scale, shake up, filter, get subsequent filtrate, promptly get need testing solution; Precision takes by weighing through the Gardenoside reference substance 10mg of phosphorus pentoxide drying under reduced pressure more than 12 hours, puts in the 25ml measuring bottle, adds dissolve with methanol to scale, shakes up, precision is measured 1ml, puts in the 10ml measuring bottle, adds methyl alcohol to scale, shake up, contain Gardenoside 40 μ g among every 1ml, promptly get reference substance solution; With octadecylsilane chemically bonded silica is filling agent, acetonitrile-water be 5: 95 for moving phase, detect wavelength 238nm, number of theoretical plate calculates by the Gardenoside peak should be not less than 2000; According to high effective liquid chromatography for measuring, accurate respectively reference substance solution and each 10 μ l injection liquid chromatograph of need testing solution drawn measured, that is, every 1ml formulation for clearing wind-heat of children contains cape jasmine with Gardenoside C ₁₇H ₂₄O ₁₀Meter must not be less than 0.16mg.

Differentiate:

(1) gets formulation for clearing wind-heat of children 50ml, add watery hydrochloric acid adjust pH to 1, put in the separating funnel, extract 2 times with solvent ether, 90 ℃ of sherwood oils or methenyl choloride, each 30ml, discard solvent ether, 90 ℃ of sherwood oils or methenyl choloride liquid, use ethyl acetate extraction again three times, each 20ml, combined ethyl acetate liquid, water bath method, residue add methyl alcohol 5ml dissolving, as need testing solution; Other gets burdock glycosides reference substance, adds methyl alcohol and makes the solvent that every 1ml contains 5mg, in contrast product solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw reference substance solution 5 μ l, need testing solution 10 μ l, put respectively on same silica gel g thin-layer plate, with methenyl choloride-methanol-water be 50: 1: 5 be developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing at 105 ℃; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color;

(2) get formulation for clearing wind-heat of children 10ml, add watery hydrochloric acid adjust pH to 2, put in the separating funnel, extract 2 times with solvent ether, 60 ℃ of sherwood oils or methenyl choloride, each 10ml, discard solvent ether, 60 ℃ of sherwood oils or methenyl choloride liquid, use ethyl acetate extraction again three times, each 20ml, combined ethyl acetate liquid, water bath method, residue add methyl alcohol 1ml dissolving, as need testing solution; Other gets the scutelloside reference substance, adds methyl alcohol and makes solution that every ml contains 0.5mg product solution in contrast; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw reference substance solution 5 μ l, need testing solution 2 μ l, put respectively on same silica gel g thin-layer plate, with ethyl acetate-butanone-formic acid-water is 3: 5: 0.1: 5 is developping agent, launch, take out, dry, spray is with 1% ferric trichloride ethanolic solution; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color;

(3) get formulation for clearing wind-heat of children 50ml, inject in the D101 type macroporous adsorptive resins of the internal diameter 1.5cm, the high 25cm of post that have handled well, use the speed wash-out of the distilled water of 500ml earlier, discard eluent with 5ml/min, use 95% ethanol elution again, the collection eluent, shakes up to scale in the 100ml measuring bottle, filters, get subsequent filtrate 60ml and put evaporating dish, evaporate to dryness, residue add methyl alcohol 4ml dissolving, as need testing solution; Other gets the Gardenoside reference substance, adds methyl alcohol and makes the solution that every 1ml contains 4mg, in contrast product solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw need testing solution 10 μ l, reference substance solution 5 μ l, put respectively on same silica gel g thin-layer plate, with methenyl choloride-methyl alcohol-acetonitrile-ammoniacal liquor is 10: 1: 1: 0.1 is developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and 105 ℃ to be heated to the spot colour developing clear; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;

(4) get formulation for clearing wind-heat of children 50ml, with ammonia solution adjust pH to 10, with solvent ether, 90 ℃ of sherwood oils or chloroform extraction 2 times, each 20ml divides and gets solvent ether, 90 ℃ of sherwood oils or methenyl choloride liquid, evaporate to dryness, residue adds methyl alcohol or ethanol 5ml makes dissolving, as need testing solution; Other gets capsule of weeping forsythia control medicinal material 5g and adds water 60ml, decocts 60 minutes, filters, and filtrate is concentrated into 50ml, prepares medicinal material solution in contrast with method; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography; Draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with methenyl choloride-ethyl acetate-methyl alcohol be 10: 2: 5 be developping agent, launch, take out, dry, spray is with 5% ethanol solution of sulfuric acid, 105 ℃ be heated to spot develop the color clear; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of identical color;

(5) get formulation for clearing wind-heat of children 10ml, add water 5ml, shake up, add watery hydrochloric acid and regulate pH value to 2, centrifugal 10 minutes, divide and get supernatant solubilizer ether, 60 ℃ of sherwood oils or methenyl choloride, ethyl acetate 10ml jolting extraction 2 times, divide and to get solvent ether, 60 ℃ of sherwood oils or methenyl choloride, acetic acid ethyl fluid, evaporate to dryness, residue solubilizer ether, 60 ℃ of sherwood oils or methenyl choloride, ethyl acetate 1ml make dissolving, as need testing solution; Other gets the chlorogenic acid reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.1mg, in contrast product solution; According to the test of an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw each 2 μ l of above-mentioned two kinds of solution, put respectively on same polyamide film, with ethyl acetate-methyl alcohol-formic acid be 5: 5: 0.1 be developping agent, launch, take out, dry, put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color;

(6) get formulation for clearing wind-heat of children 50ml, use solvent ether, 90 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol jolting are extracted 3 times, each 50ml, merge solvent ether, 90 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol liquid, wash with 2% sodium hydroxide solution 30ml, use solvent ether again, 90 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol washing 2 times, each 30ml, discard washing lotion, solvent ether, 90 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol solution evaporate to dryness, residue adds methyl alcohol 5ml makes dissolving, as need testing solution; Other gets radix bupleuri control medicinal material 5g, adds water 100ml, heats little boiling 5 hours, and filtrate is shone medicinal material solution in pairs with legal system; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw above-mentioned need testing solution 10 μ l, control medicinal material solution 5 μ l, put respectively on same silica gel g thin-layer plate, with methenyl choloride-methanol-water be 50: 5: 10 be developping agent, launch, taking-up is dried, and spray is with 40% sulfuric acid solution of 2% paradime thylaminobenzaldehyde, and 100 ℃ to be heated to the spot colour developing clear; Put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence principal spot of same color;

(7) get formulation for clearing wind-heat of children 10ml, put in the separating funnel, solubilizer ether, 60 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol 10ml joltings are extracted 1 time, divide and get solvent ether, 60 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol liquid, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the aurantiin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.1mg, in contrast product solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw above-mentioned need testing solution 10 μ l, reference substance solution 5 μ l, put respectively on same silica gel g thin-layer plate, with ethyl acetate-butanone-formic acid-water is 2: 5: 0.1: 5 is developping agent, launch, taking-up is dried, and spray is with 2% aluminium choride ethanolic solution, 105 ℃ of heating several minutes, put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color.

Assay:

Precision is measured formulation for clearing wind-heat of children 50ml, inject in the D101 type macroporous adsorptive resins of the internal diameter 1.5cm, the high 25cm of post that have handled well, use the speed wash-out of the distilled water of 500ml earlier with 5ml/min, discard eluent, use 95% ethanol elution again, collect eluent in the 100ml measuring bottle to scale, shake up, filter, get subsequent filtrate, promptly get need testing solution; Precision takes by weighing through the Gardenoside reference substance 10mg of phosphorus pentoxide drying under reduced pressure more than 12 hours, puts in the 25ml measuring bottle, adds dissolve with methanol to scale, shakes up, precision is measured 1ml, puts in the 10ml measuring bottle, adds methyl alcohol to scale, shake up, contain Gardenoside 40 μ g among every 1ml, promptly get reference substance solution; With octadecylsilane chemically bonded silica is filling agent, acetonitrile-water be 30: 70 for moving phase, detect wavelength 238nm, number of theoretical plate calculates by the Gardenoside peak should be not less than 2000; According to high effective liquid chromatography for measuring, accurate respectively reference substance solution and each 10 μ l injection liquid chromatograph of need testing solution drawn measured, that is, every 1ml formulation for clearing wind-heat of children contains cape jasmine with Gardenoside C ₁₇H ₂₄O ₁₀Meter must not be less than 0.16mg.

Differentiate:

(1) gets formulation for clearing wind-heat of children 25ml, add watery hydrochloric acid adjust pH to 1, put in the separating funnel, extract 2 times with solvent ether, 70 ℃ of sherwood oils or methenyl choloride, each 20ml, discard solvent ether, 70 ℃ of sherwood oils or methenyl choloride liquid, use ethyl acetate extraction again three times, each 20ml, combined ethyl acetate liquid, water bath method, residue add methyl alcohol 3ml dissolving, as need testing solution; Other gets burdock glycosides reference substance, adds methyl alcohol and makes the solvent that every 1ml contains 3mg, in contrast product solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw reference substance solution 5 μ l, need testing solution 10 μ l, put respectively on same silica gel g thin-layer plate, with methenyl choloride-methanol-water be 30: 5: 1 be developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing at 105 ℃; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color;

(2) get formulation for clearing wind-heat of children 20ml, add watery hydrochloric acid adjust pH to 2, put in the separating funnel, extract 2 times with solvent ether, 80 ℃ of sherwood oils or methenyl choloride, each 20ml, discard solvent ether, 80 ℃ of sherwood oils or methenyl choloride liquid, use ethyl acetate extraction again three times, each 20ml, combined ethyl acetate liquid, water bath method, residue add methyl alcohol 3ml dissolving, as need testing solution; Other gets the scutelloside reference substance, adds methyl alcohol and makes solution that every ml contains 3mg product solution in contrast; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw reference substance solution 5 μ l, need testing solution 2 μ l, put respectively on same silica gel g thin-layer plate, with ethyl acetate-butanone-formic acid-water is 4: 3: 4: 2 is developping agent, launch, take out, dry, spray is with 1% ferric trichloride ethanolic solution; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color;

(3) get formulation for clearing wind-heat of children 30ml, inject in the D101 type macroporous adsorptive resins of the internal diameter 1.5cm, the high 25cm of post that have handled well, use the speed wash-out of the distilled water of 300ml earlier, discard eluent with 3ml/min, use 75% ethanol elution again, the collection eluent, shakes up to scale in the 100ml measuring bottle, filters, get subsequent filtrate 20ml and put evaporating dish, evaporate to dryness, residue add methyl alcohol 1ml dissolving, as need testing solution; Other gets the Gardenoside reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5mg, in contrast product solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw need testing solution 10 μ l, reference substance solution 5 μ l, put respectively on same silica gel g thin-layer plate, with methenyl choloride-methyl alcohol-acetonitrile-ammoniacal liquor is 1: 5: 0.1: 1 is developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and 105 ℃ to be heated to the spot colour developing clear; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;

(4) get formulation for clearing wind-heat of children 30ml, with ammonia solution adjust pH to 10, with solvent ether, 80 ℃ of sherwood oils or chloroform extraction 2 times, each 20ml divides and gets solvent ether, 80 ℃ of sherwood oils or methenyl choloride liquid, evaporate to dryness, residue adds methyl alcohol or ethanol 2ml makes dissolving, as need testing solution; Other gets capsule of weeping forsythia control medicinal material 3g and adds water 70ml, decocts 40 minutes, filters, and filtrate is concentrated into 30ml, prepares medicinal material solution in contrast with method; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography; Draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with methenyl choloride-ethyl acetate-methyl alcohol be 50: 8: 2 be developping agent, launch, take out, dry, spray is with 5% ethanol solution of sulfuric acid, 105 ℃ be heated to spot develop the color clear; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of identical color;

(5) get formulation for clearing wind-heat of children 20ml, add water 10ml, shake up, add watery hydrochloric acid and regulate pH value to 1, centrifugal 10 minutes, divide and get supernatant solubilizer ether, 70 ℃ of sherwood oils or methenyl choloride, ethyl acetate 20ml jolting extraction 3 times, divide and to get solvent ether, 70 ℃ of sherwood oils or methenyl choloride, acetic acid ethyl fluid, evaporate to dryness, residue solubilizer ether, 70 ℃ of sherwood oils or methenyl choloride, ethyl acetate 3ml make dissolving, as need testing solution; Other gets the chlorogenic acid reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5mg, in contrast product solution; According to the test of an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw each 2 μ l of above-mentioned two kinds of solution, put respectively on same polyamide film, with ethyl acetate-methyl alcohol-formic acid be 6: 4: 3 be developping agent, launch, take out, dry, put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color;

(6) get formulation for clearing wind-heat of children 10ml, use solvent ether, 60 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol jolting are extracted 1 time, each 10ml, merge solvent ether, 60 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol liquid, wash with 2% sodium hydroxide solution 10ml, use solvent ether again, 60 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol washing 2 times, each 30ml, discard washing lotion, solvent ether, 60 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol solution evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other gets radix bupleuri control medicinal material 1g, adds water 50ml, heats little boiling 1 hour, and filtrate is shone medicinal material solution in pairs with legal system; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw above-mentioned need testing solution 10 μ l, control medicinal material solution 5 μ l, put respectively on same silica gel g thin-layer plate, with methenyl choloride-methanol-water be 10: 30: 10 be developping agent, launch, taking-up is dried, and spray is with 40% sulfuric acid solution of 2% paradime thylaminobenzaldehyde, and 60 ℃ to be heated to the spot colour developing clear; Put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence principal spot of same color;

(7) get formulation for clearing wind-heat of children 30ml, put in the separating funnel, solubilizer ether, 75 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol 20ml joltings are extracted 2 times, divide and get solvent ether, 75 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol liquid, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the aurantiin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5mg, in contrast product solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw above-mentioned need testing solution 10 μ l, reference substance solution 5 μ l, put respectively on same silica gel g thin-layer plate, with ethyl acetate-butanone-formic acid-water is 6: 2: 2: 4 is developping agent, launch, taking-up is dried, and spray is with 2% aluminium choride ethanolic solution, 105 ℃ of heating several minutes, put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color.

Assay:

Precision is measured formulation for clearing wind-heat of children 30ml, inject in the D101 type macroporous adsorptive resins of the internal diameter 1.5cm, the high 25cm of post that have handled well, use the speed wash-out of the distilled water of 300ml earlier with 3ml/min, discard eluent, use 75% ethanol elution again, collect eluent in the 100ml measuring bottle to scale, shake up, filter, get subsequent filtrate, promptly get need testing solution; Precision takes by weighing through the Gardenoside reference substance 10mg of phosphorus pentoxide drying under reduced pressure more than 12 hours, puts in the 25ml measuring bottle, adds dissolve with methanol to scale, shakes up, precision is measured 1ml, puts in the 10ml measuring bottle, adds methyl alcohol to scale, shake up, contain Gardenoside 40 μ g among every 1ml, promptly get reference substance solution; With octadecylsilane chemically bonded silica is filling agent, acetonitrile-water be 20: 85 for moving phase, detect wavelength 238nm, number of theoretical plate calculates by the Gardenoside peak should be not less than 2000; According to high effective liquid chromatography for measuring, accurate respectively reference substance solution and each 10 μ l injection liquid chromatograph of need testing solution drawn measured, that is, every 1ml formulation for clearing wind-heat of children contains cape jasmine with Gardenoside C ₁₇H ₂₄O ₁₀Meter must not be less than 0.16mg.

Existing detection method is can national standard improve requirement according to state-promulgated pharmacopoeia, mainly set about revising, replenish, improve quality standard detecting method from simple to operate, saving detection cost, reduction reagent toxicity, the detection method of formulation for clearing wind-heat of children has following advantage:

(1) as extracting solvent, toxicity is big with benzene for former detection method, and the spot that detects great burdock achene is more, is difficult for judging.Existing method improvement the extraction solvent use " ether, 70 ℃ of sherwood oils or methenyl choloride " instead, detect burdock glycosides spot, operation, judge simply, the Rf value is moderate.

(2) the former method of inspection is washed with ether, uses ethyl acetate extraction again.Use instead now and differentiate a need testing solution point sample.Former method of inspection point sample on polyamide film now changes silica G plate point sample into.Detect the scutelloside clear spot, simple to operate, save cost.

(3) method of inspection all is to contain formulations prepared from solutions point sample under the quantifier.Former content need testing solution extracts uses extracting n-butyl alcohol 6 times, and not only method complexity but also reagent dosage are big, now change to such an extent that content method was both saved time, and save reagent and cost again.

(4) the former method of inspection is that the direct sample product concentrate the back point sample, and after launching to develop the color, the blurring background is dark.Change into now behind the soup adjust pH with solvent ether, 80 ℃ of sherwood oils or chloroform extraction, detect capsule of weeping forsythia clear spot, the Rf value is moderate.Experiment and embodiment are used to further specify but are not limited to the present invention below

Experimental example 1 assay

Gardenoside is the characteristic effective constituent of cape jasmine, and primary standard is a thin layer chromatography scanning, and existing revision is high performance liquid chromatography.

1, instrument and reagent

Instrument: day island proper Tianjin HPLC chromatograph LC-10ATvp pump, SPD-10ATvp UV-detector, N2010 Zhejiang University chromatographic data workstation.Chromatographic column: Dalian Yi Teli, specification 250nm * 4.6nm * 10 μ m.

Reference substance: Gardenoside uses dry use more than 12 hours in the preposition phosphorus pentoxide vacuum drying apparatus for the 110749-200512 that Nat'l Pharmaceutical ﹠ Biological Products Control Institute provides supplies the assay medicine.

Reagent: acetonitrile is a chromatorgaphy reagent, and water is redistilled water, and it is pure that other reagent is analysis.

2, chromatographic condition

(1) choice of experimental conditions

Chromatographic column: HypersilCl8, specification 250nm * 4.6nm * 10 μ m; Moving phase: 12: 88 acetonitrile-water; Flow velocity 1.0ml/min; Column temperature is a room temperature; Detect wavelength 238nm; Sensitivity 0.01AUFS.Under selected condition, but other component chromatographic peak baseline separation in Gardenoside and the sample.Gardenoside is adjacent the degree of separation of chromatographic peak greater than 1.5, and Gardenoside peak theoretical cam curve is more than 2000, and holder tail factor T is 0.99.

The selection of maximum absorption wavelength: reference substance solution is carried out the uv scan maximum absorption wavelength and is positioned at the 237.6nm place, so the Liquid Detection wavelength is decided to be 238nm.(see figure 1)

The selection of (2) pre-treatment condition

1. column length is definite: under the identical condition of other conditions, with the resin column test of 15cm, 20cm, 25cm, 30cm, the impurity of the post of 25cm absorption as a result is the most thorough respectively, and the time spent is also less, collection of illustrative plates peak the best.

2. the wash-out of variable concentrations relatively: under the identical condition of other conditions, use 60%, 70%, 80% ethanol elution respectively, survey its content respectively.The results are shown in Table 1:

Table 1: record the content data table behind the variable concentrations wash-out

Wash-out	60％	70％	80％
				Content	0.28mg/ml	0.32mg/ml	0.31mg/ml

Test shows, is the sample eluting solvent so adopt 70% ethanol.

3. 70% ethanol elution volume is definite: under the identical situation of other conditions, use 100ml, 150ml wash-out respectively, survey its content respectively, the results are shown in Table 2:

Table 2: different elution volumes record the content data table

Presentation of results 150ml wash-out content has not had increase than 100ml wash-out content, so get final product with 100ml 70% ethanol elution.

For understanding fully the precise volumes of wash-out, we are divided into preceding 30ml with the 100ml elution volume under the prerequisite of same resin column, middle 30ml, and back 40ml collects eluent respectively for three sections, and injecting chromatograph is measured area, the results are shown in Table 3:

Table 3: different elution volumes record the peak area tables of data

Volume	Preceding 30ml	Middle 30ml	Back 40ml
				Peak area	130935	312512.9	3027.6

The Gardenoside of 30ml wash-out was maximum in the middle of the result showed.

3, methodology checking

(1) accuracy

Precision is measured the same lot number 1020 sample 5ml of known Gardenoside content, accurate respectively 0.1072mg/ml Gardenoside reference substance solution 1.0ml, 1.5ml, the 1.7ml of adding presses the said determination condition and measures, and following column count formula, calculate recovery rate, test findings sees Table 4

Table 4: sample recovery rate test findings (n=9)

Test shows: this law has the good recovery.

(2) precision

Got same lot number sample 6 minutes, precision is measured, and prepares need testing solution by above-mentioned condition, and the peak area value of survey also calculates content.RSD＜2% (n=6) the results are shown in Table 5

Table 5: sample reappearance test

Instrument precision

The accurate reference substance solution of drawing repeats sample introduction 5 times, and the RSD of Gardenoside peak area value＜2% (n=5) the results are shown in Table 6

Table 6: instrument precision experiment

Test shows that instrument precision is good.

(3) linearity

Precision is measured 0.04288mg/ml Gardenoside reference substance solution; Accurate respectively reference substance solution 3 μ l, 6 μ l, 9 μ l, the 12 μ l of drawing, 15 μ l inject liquid chromatograph, measure peak area by above-mentioned chromatographic condition, are horizontal ordinate with the Gardenoside micrograms, peak area is an ordinate, gets a straight line, calculates regression equation, Y=-2.58536 * 10 ^-5X+1.36031 * 10 ^-9, r=0.99928, n=5.The result shows that Gardenoside is good in 0.12864 μ g～0.6432 μ g scope internal linear relation.(see figure 2)

(4) specificity

Ratio in the prescription taste of traditional Chinese medicine, take by weighing the group's medicine that does not contain cape jasmine, make negative control by preparation process, prepare the negative control solution that lacks cape jasmine by the need testing solution preparation method again, draw reference substance solution, need testing solution, each 20 μ l of negative control solution, injecting chromatograph is measured in accordance with the law respectively, the identical retention time with the Gardenoside reference substance of negative control solution place does not show chromatographic peak as a result, so think noiseless.(seeing Fig. 3,4,5)

(5) durability

Sample thief prepares lot number 1020 need testing solutions by above-mentioned condition, injects liquid chromatograph, measures peak area, measures once in 0,4,8,16 hour then, and X=445496 RSD=1.45% the results are shown in Table 7

Table 7: durability experiment

The result shows that need testing solution is basicly stable at 16 hours.

4, sample and raw material cape jasmine assay

Sample thief prepares need testing solution in accordance with the law, draws each 20 μ l of reference substance solution and need testing solution and injects hplc determination peak area, calculation sample Gardenoside content.See Table 8

Table 8: sample size measurement result

Lot number	Gardenoside content (mg/ml)
		050605	0.35
050606	0.28
		050607	0.30
050608	0.29
		050609	0.28
050816	0.18
		050817	0.17
050819	0.18
		050926	0.30
050927	0.35

Raw material cape jasmine assay the results are shown in Table 9

Table 9: raw material cape jasmine assay result

The raw material numbering	1 ^#	2 ^#	3 ^#	4 ^#
					Gardenoside content (%)	3.1	1.82	2.5	3.6

The cape jasmine material content of 4 numberings of root a tree name is learnt, because of the raw material sources difference, raw material cape jasmine content difference is bigger, thereby causes the fluctuation of finished product content bigger, 10 batches of finished product content data of root a tree name are defined as content limit: per 1 milliliter of formulation for clearing wind-heat of children contains cape jasmine with Gardenoside C ₁₇H ₂₄O ₁₀Meter must not be less than 0.16mg.

Description of drawings:

Fig. 1: uv scan figure;

Fig. 2: Gardenoside linear relationship chart;

Fig. 3: reference substance solution uv scan figure

Fig. 4: need testing solution uv scan figure;

Fig. 5: negative control solution uv scan figure

Following embodiment all can realize the effect of above-mentioned experimental example.

Embodiment

Embodiment 1: the detection method of formulation for clearing wind-heat of children

Differentiate:

(1) gets formulation for clearing wind-heat of children 10ml, add watery hydrochloric acid adjust pH to 2, put in the separating funnel, extract 2 times with solvent ether, each 10ml, discard solvent ether liquid, use ethyl acetate extraction again three times, each 20ml, combined ethyl acetate liquid, water bath method, residue add methyl alcohol 1ml dissolving, as need testing solution; Other gets burdock glycosides reference substance, adds methyl alcohol and makes the solvent that every 1ml contains 1mg, in contrast product solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw reference substance solution 5 μ l, need testing solution 10 μ l, put respectively on same silica gel g thin-layer plate, with methenyl choloride-methanol-water be 10: 10: 1 be developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing at 105 ℃; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color;

(2) get formulation for clearing wind-heat of children 50ml, add watery hydrochloric acid adjust pH to 1, put in the separating funnel, use 90 ℃ of Petroleum ether extraction of solvent 2 times, each 30ml, discard 90 ℃ of sherwood oil liquid of solvent, use ethyl acetate extraction again three times, each 20ml, combined ethyl acetate liquid, water bath method, residue add methyl alcohol 5ml dissolving, as need testing solution; Other gets the scutelloside reference substance, adds methyl alcohol and makes solution that every ml contains 5mg product solution in contrast; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw reference substance solution 5 μ l, need testing solution 2 μ l, put respectively on same silica gel g thin-layer plate, with ethyl acetate-butanone-formic acid-water is 10: 1: 5: 0.1 is developping agent, launch, take out, dry, spray is with 1% ferric trichloride ethanolic solution; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color;

(4) get formulation for clearing wind-heat of children 20ml, with ammonia solution adjust pH to 10, use solvent chloroform extraction 2 times, each 20ml divides and gets solvent methenyl choloride liquid, and evaporate to dryness, residue add methyl alcohol or ethanol 2ml makes dissolving, as need testing solution; Other gets capsule of weeping forsythia control medicinal material 2g and adds water 90ml, decocts 20 minutes, filters, and filtrate is concentrated into 20ml, prepares medicinal material solution in contrast with method; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography; Draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with methenyl choloride-ethyl acetate-methyl alcohol be 3: 9: 3 be developping agent, launch, take out, dry, spray is with 5% ethanol solution of sulfuric acid, 105 ℃ be heated to spot develop the color clear; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of identical color;

(5) get formulation for clearing wind-heat of children 40ml, add water 20ml, shake up, add watery hydrochloric acid and regulate pH value to 1, centrifugal 10 minutes, divide and get supernatant solubilizer ether 30ml jolting extraction 3 times, divide and get solvent ether liquid, evaporate to dryness, residue solubilizer ether 4ml makes dissolving, as need testing solution; Other gets the chlorogenic acid reference substance, adds methyl alcohol and makes the solution that every 1ml contains 1mg, in contrast product solution; According to the test of an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw each 2 μ l of above-mentioned two kinds of solution, put respectively on same polyamide film, with ethyl acetate-methyl alcohol-formic acid be 10: 0.5: 4 be developping agent, launch, take out, dry, put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color;

(6) get formulation for clearing wind-heat of children 25ml, extract 2 times with the jolting of solvent water-saturated n-butanol, each 25ml merges solvent water-saturated n-butanol liquid, washs with 2% sodium hydroxide solution 20ml, again with solvent water-saturated n-butanol washing 2 times, each 30ml discards washing lotion, the saturated butanol solution evaporate to dryness of aqueous solvent, residue adds methyl alcohol 5ml makes dissolving, as need testing solution; Other gets radix bupleuri control medicinal material 5g, adds water 70ml, heats little boiling 2 hours, and filtrate is shone medicinal material solution in pairs with legal system; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw above-mentioned need testing solution 10 μ l, control medicinal material solution 5 μ l, put respectively on same silica gel g thin-layer plate, with methenyl choloride-methanol-water be 50: 15: 5 be developping agent, launch, taking-up is dried, and spray is with 40% sulfuric acid solution of 2% paradime thylaminobenzaldehyde, and 60 ℃ to be heated to the spot colour developing clear; Put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence principal spot of same color;

(7) get formulation for clearing wind-heat of children 50ml, put in the separating funnel, solubilizer ethyl acetate 30ml jolting is extracted 3 times, divides and gets solvent ethyl acetate liquid, and evaporate to dryness, residue add methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the aurantiin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 1mg, in contrast product solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw above-mentioned need testing solution 10 μ l, reference substance solution 5 μ l, put respectively on same silica gel g thin-layer plate, with ethyl acetate-butanone-formic acid-water is 10: 1: 3: 0.1 is developping agent, launch, taking-up is dried, and spray is with 2% aluminium choride ethanolic solution, 105 ℃ of heating several minutes, put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color.

Assay:

Embodiment 2: the detection method of formulation for clearing wind-heat of children

Assay:

Embodiment 3: the detection method of formulation for clearing wind-heat of children

Differentiate:

(1) gets formulation for clearing wind-heat of children 50ml, add watery hydrochloric acid adjust pH to 1, put in the separating funnel, use 90 ℃ of Petroleum ether extraction of solvent 2 times, each 30ml, discard 90 ℃ of sherwood oil liquid of solvent, use ethyl acetate extraction again three times, each 20ml, combined ethyl acetate liquid, water bath method, residue add methyl alcohol 5ml dissolving, as need testing solution; Other gets burdock glycosides reference substance, adds methyl alcohol and makes the solvent that every 1ml contains 5mg, in contrast product solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw reference substance solution 5 μ l, need testing solution 10 μ l, put respectively on same silica gel g thin-layer plate, with methenyl choloride-methanol-water be 50: 1: 5 be developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing at 105 ℃; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color;

(2) get formulation for clearing wind-heat of children 10ml, add watery hydrochloric acid adjust pH to 2, put in the separating funnel, extract 2 times with the solvent methenyl choloride, each 10ml, discard solvent methenyl choloride liquid, use ethyl acetate extraction again three times, each 20ml, combined ethyl acetate liquid, water bath method, residue add methyl alcohol 1ml dissolving, as need testing solution; Other gets the scutelloside reference substance, adds methyl alcohol and makes solution that every ml contains 0.5mg product solution in contrast; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw reference substance solution 5 μ l, need testing solution 2 μ l, put respectively on same silica gel g thin-layer plate, with ethyl acetate-butanone-formic acid-water is 3: 5: 0.1: 5 is developping agent, launch, take out, dry, spray is with 1% ferric trichloride ethanolic solution; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color;

(4) get formulation for clearing wind-heat of children 50ml, with ammonia solution adjust pH to 10, use 90 ℃ of petroleum ether extractions of solvent 2 times, each 20ml divides and gets 90 ℃ of sherwood oil liquid of solvent, and evaporate to dryness, residue add methyl alcohol 5ml makes dissolving, as need testing solution; Other gets capsule of weeping forsythia control medicinal material 5g and adds water 60ml, decocts 60 minutes, filters, and filtrate is concentrated into 50ml, prepares medicinal material solution in contrast with method; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography; Draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with methenyl choloride-ethyl acetate-methyl alcohol be 10: 2: 5 be developping agent, launch, take out, dry, spray is with 5% ethanol solution of sulfuric acid, 105 ℃ be heated to spot develop the color clear; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of identical color;

(5) get formulation for clearing wind-heat of children 10ml, add water 5ml, shake up, add watery hydrochloric acid and regulate pH value to 2, centrifugal 10 minutes, divide and get supernatant solubilizer ethyl acetate 10ml jolting extraction 2 times, divide and get solvent ethyl acetate liquid, evaporate to dryness, residue solubilizer ethyl acetate 1ml makes dissolving, as need testing solution; Other gets the chlorogenic acid reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.1mg, in contrast product solution; According to the test of an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw each 2 μ l of above-mentioned two kinds of solution, put respectively on same polyamide film, with ethyl acetate-methyl alcohol-formic acid be 5: 5: 0.1 be developping agent, launch, take out, dry, put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color;

(6) get formulation for clearing wind-heat of children 50ml, extract 3 times with the jolting of solvent water-saturated n-butanol, each 50ml merges solvent water-saturated n-butanol liquid, washs with 2% sodium hydroxide solution 30ml, again with solvent water-saturated n-butanol washing 2 times, each 30ml discards washing lotion, the saturated butanol solution evaporate to dryness of aqueous solvent, residue adds methyl alcohol 5ml makes dissolving, as need testing solution; Other gets radix bupleuri control medicinal material 5g, adds water 100ml, heats little boiling 5 hours, and filtrate is shone medicinal material solution in pairs with legal system; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw above-mentioned need testing solution 10 μ l, control medicinal material solution 5 μ l, put respectively on same silica gel g thin-layer plate, with methenyl choloride-methanol-water be 50: 5: 10 be developping agent, launch, taking-up is dried, and spray is with 40% sulfuric acid solution of 2% paradime thylaminobenzaldehyde, and 100 ℃ to be heated to the spot colour developing clear; Put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence principal spot of same color;

(7) get formulation for clearing wind-heat of children 10ml, put in the separating funnel, solubilizer methenyl choloride 10ml jolting is extracted 1 time, divides and gets solvent methenyl choloride liquid, and evaporate to dryness, residue add methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the aurantiin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.1mg, in contrast product solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw above-mentioned need testing solution 10 μ l, reference substance solution 5 μ l, put respectively on same silica gel g thin-layer plate, with ethyl acetate-butanone-formic acid-water is 2: 5: 0.1: 5 is developping agent, launch, taking-up is dried, and spray is with 2% aluminium choride ethanolic solution, 105 ℃ of heating several minutes, put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color.

Embodiment 4: the detection method of formulation for clearing wind-heat of children

Differentiate:

(1) gets formulation for clearing wind-heat of children 20ml, add water 10ml, shake up, add watery hydrochloric acid and regulate pH value to 1, centrifugal 10 minutes, divide and get supernatant solubilizer ethyl acetate 20ml jolting extraction 3 times, divide and get solvent ethyl acetate liquid, evaporate to dryness, residue solubilizer ethyl acetate 3ml makes dissolving, as need testing solution; Other gets the chlorogenic acid reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5mg, in contrast product solution; According to the test of an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw each 2 μ l of above-mentioned two kinds of solution, put respectively on same polyamide film, with ethyl acetate-methyl alcohol-formic acid be 6: 4: 3 be developping agent, launch, take out, dry, put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color;

(2) get formulation for clearing wind-heat of children 10ml, extract 1 time with the solvent ether jolting, each 10ml merges solvent ether liquid, washs with 2% sodium hydroxide solution 10ml, again with solvent ether washing 2 times, each 30ml discards washing lotion, solvent ether solution evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other gets radix bupleuri control medicinal material 1g, adds water 50ml, heats little boiling 1 hour, and filtrate is shone medicinal material solution in pairs with legal system; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw above-mentioned need testing solution 10 μ l, control medicinal material solution 5 μ l, put respectively on same silica gel g thin-layer plate, with methenyl choloride-methanol-water be 10: 30: 10 be developping agent, launch, taking-up is dried, and spray is with 40% sulfuric acid solution of 2% paradime thylaminobenzaldehyde, and 60 ℃ to be heated to the spot colour developing clear; Put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence principal spot of same color;

(3) get formulation for clearing wind-heat of children 30ml, put in the separating funnel, 75 ℃ of sherwood oil 20ml of solubilizer jolting is extracted 2 times, divides and gets 75 ℃ of sherwood oil liquid of solvent, and evaporate to dryness, residue add methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the aurantiin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5mg, in contrast product solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw above-mentioned need testing solution 10 μ l, reference substance solution 5 μ l, put respectively on same silica gel g thin-layer plate, with ethyl acetate-butanone-formic acid-water is 6: 2: 2: 4 is developping agent, launch, taking-up is dried, and spray is with 2% aluminium choride ethanolic solution, 105 ℃ of heating several minutes, put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color.

Assay:

Embodiment 5: the detection method of formulation for clearing wind-heat of children

Differentiate:

Get formulation for clearing wind-heat of children 50ml, put in the separating funnel, solubilizer methenyl choloride 30ml jolting is extracted 3 times, divides and gets solvent methenyl choloride liquid, and evaporate to dryness, residue add methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the aurantiin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 1mg, in contrast product solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw above-mentioned need testing solution 10 μ l, reference substance solution 5 μ l, put respectively on same silica gel g thin-layer plate, with ethyl acetate-butanone-formic acid-water is 10: 1: 3: 0.1 is developping agent, launch, taking-up is dried, and spray is with 2% aluminium choride ethanolic solution, 105 ℃ of heating several minutes, put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color.

Assay:

Claims

1. the detection method of a formulation for clearing wind-heat of children is characterized in that this method comprises one or more in following discriminating and/or the content assaying method:

Differentiate:

(3) get formulation for clearing wind-heat of children 10-50ml, inject in the D101 type macroporous adsorptive resins of the internal diameter 1.5cm, the high 25cm of post that have handled well, use the speed wash-out of the distilled water of 100-500ml earlier, discard eluent with 1-5ml/min, use the ethanol elution of 50-95% again, the collection eluent, shakes up to scale in the 100ml measuring bottle, filters, get subsequent filtrate 20-80ml and put evaporating dish, evaporate to dryness, residue add methyl alcohol 1-5ml dissolving, as need testing solution; Other gets the Gardenoside reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5-5mg, in contrast product solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw need testing solution 10 μ l, reference substance solution 5 μ l, put respectively on same silica gel g thin-layer plate, with methenyl choloride-methyl alcohol-acetonitrile-ammoniacal liquor is that 1-10: 1-5: 0.1-1: 0.1-1 is a developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and 105 ℃ to be heated to the spot colour developing clear; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;

(4) get formulation for clearing wind-heat of children 10-50ml, with ammonia solution adjust pH to 10, with solvent ether, 60～90 ℃ of sherwood oils or chloroform extraction 1-3 time, each 10-20ml, divide and to get solvent ether, 60～90 ℃ of sherwood oils or methenyl choloride liquid, evaporate to dryness, residue add methyl alcohol or ethanol 1-5ml makes dissolving, as need testing solution; Other gets capsule of weeping forsythia control medicinal material 1-5g and adds water 50-100ml, decocts 10-60 minute, filters, and filtrate is concentrated into 10-50ml, prepares medicinal material solution in contrast with method; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography; Drawing each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, is that 2-10: 2-10: 1-5 is a developping agent with methenyl choloride-ethyl acetate-methyl alcohol, launch, take out, dry, spray is with 5% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing at 105 ℃; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of identical color;

(7) get formulation for clearing wind-heat of children 10-50ml, put in the separating funnel, solubilizer ether, 60～90 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol 10-30ml joltings are extracted 1-3 time, divide and get solvent ether, 60～90 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol liquid, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the aurantiin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.1-1mg, in contrast product solution; Test according to an appendix VIB of Chinese Pharmacopoeia version in 2005 thin-layered chromatography, draw above-mentioned need testing solution 10 μ l, reference substance solution 5 μ l, put respectively on same silica gel g thin-layer plate, with ethyl acetate-butanone-formic acid-water is that 2-10: 1-5: 0.1-3: 0.1-5 is a developping agent, launch, taking-up is dried, and spray is with 2% aluminium choride ethanolic solution, 105 ℃ of heating several minutes, put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color;

Assay:

2. detection method as claimed in claim 1 is characterized in that this method is one or more in following discriminating and/or the content assaying method:

Differentiate:

(1) gets formulation for clearing wind-heat of children 10ml, add watery hydrochloric acid adjust pH to 2, put in the separating funnel, extract 2 times with solvent ether, 60 ℃ of sherwood oils or methenyl choloride, each 10ml, discard solvent ether, 60 ℃ of sherwood oils or methenyl choloride liquid, use ethyl acetate extraction again three times, each 20ml, combined ethyl acetate liquid, water bath method, residue add methyl alcohol 1ml dissolving, as need testing solution; Other gets burdock glycosides reference substance, adds methyl alcohol and makes the solvent that every 1ml contains 1mg, in contrast product solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw reference substance solution 5 μ l, need testing solution 10 μ l, put respectively on same silica gel g thin-layer plate, with methenyl choloride-methanol-water be 10: 10: 1 be developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing at 105 ℃; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color;

(7) get formulation for clearing wind-heat of children 50ml, put in the separating funnel, solubilizer ether, 90 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol 30ml joltings are extracted 3 times, divide and get solvent ether, 90 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol liquid, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the aurantiin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 1mg, in contrast product solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw above-mentioned need testing solution 10 μ l, reference substance solution 5 μ l, put respectively on same silica gel g thin-layer plate, with ethyl acetate-butanone-formic acid-water is 10: 1: 3: 0.1 is developping agent, launch, taking-up is dried, and spray is with 2% aluminium choride ethanolic solution, 105 ℃ of heating several minutes, put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color;

Assay:

3. detection method as claimed in claim 1 is characterized in that this method is one or more in following discriminating and/or the content assaying method:

Differentiate:

(2) get formulation for clearing wind-heat of children 10ml, add watery hydrochloric acid adjust pH to 2, put in the separating funnel, extract 2 times with solvent ether, 60 ℃ of sherwood oils or methenyl choloride, each 10ml, discard solvent ether, 60 ℃ of sherwood oils or methenyl choloride liquid, use ethyl acetate extraction again three times, each 20ml, combined ethyl acetate liquid, water bath method, residue add methyl alcohol 1ml dissolving, as need testing solution; Other gets the scutelloside reference substance, adds methyl alcohol and makes solution that every ml contains 0.5mg product solution in contrast; Test according to an appendix VIB of Chinese Pharmacopoeia version in 2005 thin-layered chromatography, draw reference substance solution 5 μ l, need testing solution 2 μ l, put respectively on same silica gel g thin-layer plate, with ethyl acetate-butanone-formic acid-water is 3: 5: 0.1: 5 is developping agent, launch, take out, dry, spray is with 1% ferric trichloride ethanolic solution; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color;

(7) get formulation for clearing wind-heat of children 10ml, put in the separating funnel, solubilizer ether, 60 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol 10ml joltings are extracted 1 time, divide and get solvent ether, 60 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol liquid, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the aurantiin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.1mg, in contrast product solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw above-mentioned need testing solution 10 μ l, reference substance solution 5 μ l, put respectively on same silica gel g thin-layer plate, with ethyl acetate-butanone-formic acid-water is 2: 5: 0.1: 5 is developping agent, launch, taking-up is dried, and spray is with 2% aluminium choride ethanolic solution, 105 ℃ of heating several minutes, put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color;

Assay:

4. detection method as claimed in claim 1 is characterized in that this method is one or more in following discriminating and/or the content assaying method:

Differentiate:

(7) get formulation for clearing wind-heat of children 30ml, put in the separating funnel, solubilizer ether, 75 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol 20ml joltings are extracted 2 times, divide and get solvent ether, 75 ℃ of sherwood oils, methenyl choloride, ethyl acetate or water-saturated n-butanol liquid, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the aurantiin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5mg, in contrast product solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw above-mentioned need testing solution 10 μ l, reference substance solution 5 μ l, put respectively on same silica gel g thin-layer plate, with ethyl acetate-butanone-formic acid-water is 6: 2: 2: 4 is developping agent, launch, taking-up is dried, and spray is with 2% aluminium choride ethanolic solution, 105 ℃ of heating several minutes, put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color;

Assay: